US20180256715A1 - Anti-ctla-4 blockade - Google Patents

Anti-ctla-4 blockade Download PDF

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US20180256715A1
US20180256715A1 US15/571,174 US201615571174A US2018256715A1 US 20180256715 A1 US20180256715 A1 US 20180256715A1 US 201615571174 A US201615571174 A US 201615571174A US 2018256715 A1 US2018256715 A1 US 2018256715A1
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antibody
seq
antibodies
ctla
cancer
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Robert Kirken
Georgialina Rodriguez
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University of Texas System
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University of Texas System
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
    • A61K2039/585—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation wherein the target is cancer
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the induction of an immune response requires recruitment of appropriate immune cells to the site of a foreign pathogen, e.g., a cancer cell.
  • the immune response involves the interplay of a variety of immune modulatory molecules, which not only control the induction and magnitude of the response but also the production of antibodies and/or the activation of cells that reject tissue and destroy infected and neoplastic cells.
  • tumors actively engage in immune suppression to promote their growth.
  • a variety of tumors are known to either express or to induce the expression of factors that suppress tumor-specific immune responses at the tumor site.
  • Studies have identified a number of tumor-secreted or tumor-associated immune suppressive factors—the inhibition of which may restore normal immune functions and render tumors susceptible to eradication by the host immune system.
  • These tumor-associated factors may not only act at the tumor site to suppress antitumor immunity but may also act systemically to inhibit the ability of tumor antigen encoding vaccines to induce effective antitumor immunity.
  • Neutralizing immune suppressive factors may overcome the tumor-associated immune suppression and allow the development of a productive antitumor immune response.
  • Antibody based therapeutics can be used to deplete or activate a biological system, and thus dampen the disease state directly or indirectly by stimulating or relieving suppression of the immune system.
  • One example of the benefit of blocking immune suppression to treat cancer was revealed in studies in mice where the blocking CTLA-4 (using specific antibodies such as ipilimumab) led to the rejection of implanted tumors—a blockade of the negative regulator CTLA-4 was sufficient to allow the immune system to attack cancer cells.
  • the translational significance of these findings became apparent when it was reported that the administration of an anti-human CTLA-4 antibody (ipilimumab) produced a significant increase in survival of patients with metastatic melanoma. This Phase III clinical trial result led to the subsequent FDA approval of this strategy for the treatment of melanoma. More recently, it was shown that the antibody, BMS-936558, targeting the co-inhibitory receptor PD1, which is a CTLA-4 family member, induced significant and durable responses in several types of highly refractory tumors.
  • compositions for reducing or attenuating cancer mediated immune suppression will comprise an affinity reagent that specifically binds an immune suppressive agent, e.g., CTLA-4 or co-inhibitory receptors (CIR).
  • an immune suppressive agent e.g., CTLA-4 or co-inhibitory receptors (CIR).
  • CIR co-inhibitory receptors
  • the affinity reagent neutralizes the immune suppressive agent.
  • Certain embodiments are directed to an antibody or antibody fragment that specifically binds CTLA-4 (for an example of a CTLA-4 protein having the amino acid sequence MACLGFQRHKAQLNLATRTWPCTLLFFLLFIPVFCKAMHVAQPAVVLASSRGIASFV CEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVN LTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDFLLWILAAVSS GLFFYSFLLTAVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN ((SEQ ID NO:1) see GenBank accession no. AAH74893.1)).
  • CTLA-4 for an example of a CTLA-4 protein having the amino acid sequence MACLGFQRHKAQLNLATRTWPCTLLFFLLFIPVFCKAMHVAQPAVVLASSRGIASFV CEYASPGKATEVRVTVLRQADSQ
  • the antibody or antibody fragment is a conformation specific antibody.
  • the conformation specific antibody does not bind a linear epitope with a high enough affinity to neutralize or block the activity of CTLA-4.
  • an antibody or antibody fragment can specifically bind 5, 6, 7, 8, 9, or 10 consecutive amino acids of SEQ ID NO:1.
  • an antibody or antibody fragment can specifically bind the CDR3 loop of the CTLA-4 protein (e.g., residues 134-139 of SEQ ID NO:1).
  • the antibody can specifically bind the amino acid sequence YICKVELMYPPPYYLGIGNGTQI (SEQ ID NO:2).
  • the antibody can specifically bind the amino acid sequence MYPPPY (SEQ ID NO:3).
  • an antibody or antibody fragment can specifically bind a first segment of C strands (residues 68-90 of SEQ ID NO:1) of CTLA-4.
  • the antibody specifically binds the amino acid sequence EVRVTVLRQADSQVTEVCAATYM (SEQ ID NO:4).
  • an antibody or antibody fragment can specifically bind a second segment of C strands (residues 64-78 of SEQ ID NO:1). In a further aspect an antibody specifically binds the amino acid sequence of GKATEVRVTVLRQAD (SEQ ID NO:5).
  • an antibody or antibody fragment can specifically bind a third segment of the C strand (residues 81-98 of SEQ ID NO:1).
  • an antibody binds the amino acid sequence VTEVCAATYMMGNELTFL (SEQ ID NO:6).
  • an antibody or antibody fragment binds to the segment(s) of the CTLA-4 protein that contain the amino acids that form the dimer interface.
  • the dimer interface comprises residues 51, 53, 59, 106, and 117 of SEQ ID NO:1.
  • an antibody specifically binds the amino acid sequence RGIASFVCEY (SEQ ID NO:7) or SICTGTSSGNQVNLTIQGLR (SEQ ID NO:8).
  • Certain embodiments are directed to affinity reagents that specifically bind and neutralize or block the activity of hPD1 or hBTLA.
  • an antibody or antibody fragment specifically binds human PD1, which has the amino acid sequence of MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSF SNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVR ARRNDSGTYLCGAISLAPKAQIKESLRAELRVTGTIGARRTGQPLKEDPSAVPVFSVD YGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPE DGHCSWPL (SEQ ID NO:9).
  • an antibody or antibody fragment specifically binds and neutralizes or blocks the activity of PD1.
  • an antibody or antibody fragment can bind 5, 6, 7, 8, 9, 10 or more consecutive residues of SEQ ID NO:9.
  • an antibody or antibody fragment specifically binds a segment of the C′, C′C′′, FG, or G strand of PD1.
  • an antibody binds the amino acid sequence SESFVLNWYRMSPS (SEQ ID NO:10), QTDKLAAFPEDRSQPGQDC (SEQ ID NO:11), DSGTYLCGAISLAPKAQIKES (SEQ ID NO:12), or KAQIKESLRAELRVTER (SEQ ID NO:13).
  • an antibody or antibody fragment specifically binds human BTLA, which has the amino acid sequence of MKTLPAMLGTGKLFWVFFLIPYLDIWNIHGKESCDVQLYIKRQSEHSILAGDPFELEC PVKYCANRPHVTWCKLNGTTCVKLEDRQTSWKEEKNISFFILHFEPVLPNDNGSYRC SANFQSNLIESHSTTLYVTGKQNELSDTAGREINLVDAHLKSEQTEASTRQNSQVLLS ETGIYDNDPDLCFRMQEGSEVYSNPCLEENKPGIVYASLNHSVIGLNSRLARNVKEA PTEYASICVRS (SEQ ID NO:14).
  • an antibody or antibody fragment specifically binds and neutralizes or blocks the activity of BTLA.
  • an antibody or antibody fragment can bind 5, 6, 7, 8, 9, 10 or more consecutive residues of SEQ ID NO:14.
  • an antibody or antibody fragment specifically binds a segment of the A′ strand, G′ strand, or CC′loop of BTLA.
  • an antibody binds the amino acid sequence DVQLYIKRQSEHSILA (SEQ ID NO:15), CSANFQSNLIESHS (SEQ ID NO:16), or RPHVTWCKLNGTTCVK (SEQ ID NO:17).
  • antibody or “immunoglobulin” is used to include intact antibodies and binding fragments/segments (functional fragments) thereof. “Functional fragments” of such antibodies comprise portions of intact antibodies that retain a similar antigen-binding specificity to the parent antibody molecule.
  • functional fragments can comprise at least the CDRs of either the heavy chain or light chain variable region.
  • Functional fragments can also comprise the heavy chain or light chain variable region, or sequences that are substantially similar to the heavy or light chain variable region.
  • suitable functional fragments include, without limitation, antibodies with multiple epitope specificity, bispecific antibodies, diabodies, and single-chain molecules, as well as Fab, F(ab′)2, Fd, Fabc, and Fv molecules, single chain (Sc) antibodies (also called ScFv), individual antibody light chains, individual antibody heavy chains, chimeric fusions between antibody chains and other molecules, heavy chain monomers or dimers, light chain monomers or dimers, dimers consisting of one heavy and one light chain, and the like. All antibody isotypes can be used to produce functional fragments of the antibodies herein. Functional fragments can be recombinantly or synthetically produced, with natural or unnatural nucleic acid or amino acid molecules.
  • the antibodies or functional fragments thereof of the disclosed subject matter can be generated from any species.
  • the antibodies or functional fragments thereof described herein can be labeled or otherwise conjugated to various chemical or biomolecule moieties, for example, for therapeutic or diagnostic or detection or treatment applications.
  • the moieties can be cytotoxic, for example, bacterial toxins, viral toxins, radioisotopes, and the like.
  • the moieties can be detectable labels, for example, fluorescent labels, radiolabels, biotin, and the like, which are known in the art.
  • the term “monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single epitope. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.
  • the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler, et al., Nature 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).
  • the “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991), for example.
  • human sequence antibody or “human antibody” includes antibodies having variable and constant regions (if present) derived from human germline immunoglobulin sequences.
  • the human sequence antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).
  • the term “human sequence antibody”, as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences (i.e., humanized antibodies).
  • the antibodies or functional fragments thereof described herein have binding affinities in M for their respective targets that include a dissociation constant (K D ) of less than 1 ⁇ 10 ⁇ 2 .
  • K D dissociation constant
  • the K D is less than 1 ⁇ 10 ⁇ 3 .
  • the K D is less than 1 ⁇ 10 ⁇ 4 .
  • the K D is less than 1 ⁇ 10 ⁇ 5 .
  • the K D is less than 1 ⁇ 10 ⁇ 6 , 1 ⁇ 10 ⁇ 7 , 1 ⁇ 10 ⁇ 8 , 1 ⁇ 10 ⁇ 9 , 1 ⁇ 10 ⁇ 10 , 1 ⁇ 10 ⁇ 11 , 1 ⁇ 10 ⁇ 12 , 1 ⁇ 10 ⁇ 13 , 1 ⁇ 10 ⁇ 14 , or 1 ⁇ 10 ⁇ 15 .
  • an antigen is a molecule capable of being bound by an antibody or T-cell receptor.
  • An antigen is additionally capable of inducing a humoral immune response and/or cellular immune response leading to the production of B- and/or T-lymphocytes.
  • the structural aspect of an antigen e.g., three-dimensional conformation or modification (e.g., phosphorylation), giving rise to a biological response is referred to herein as an “antigenic determinant” or “epitope.”
  • B-lymphocytes respond to foreign antigenic determinants via antibody production, whereas T-lymphocytes are the mediator of cellular immunity.
  • antigenic determinants or epitopes are those parts of an antigen that are recognized by antibodies, or in the context of an MHC, by T-cell receptors.
  • An antigenic determinant need not be a contiguous sequence or segment of protein and may include various sequences that are not immediately adjacent to one another.
  • binding moieties other than antibodies and be engineered to specifically bind to an antigen e.g., aptamers, avimers, and the like.
  • Moieties of the invention such as polypeptides, peptides, antibodies, antigens, or immunogens, may be conjugated or linked covalently or noncovalently to other moieties such as adjuvants, proteins, peptides, supports, fluorescence moieties, or labels.
  • conjugated or linked covalently or noncovalently to other moieties such as adjuvants, proteins, peptides, supports, fluorescence moieties, or labels.
  • conjugated or “immunoconjugate” is broadly used to define the operative association of one moiety with another agent and is not intended to refer solely to any type of operative association, and is particularly not limited to chemical “conjugation.”
  • the phrase “specifically binds” or “specifically immunoreactive” to a target refers to a binding reaction that is determinative of the presence of the molecule in the presence of a heterogeneous population of other biologics.
  • a specified molecule binds preferentially to a particular target and does not bind in a significant amount to other biologics present in the sample.
  • Specific binding of an antibody to a target under such conditions requires the antibody be selected for its specificity to the target.
  • a variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein.
  • solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press, 1988, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.
  • treating refers to any success or indicia of success in the attenuation or amelioration of an injury, pathology or condition, including any objective or subjective parameter such as abatement, remission, diminishing of symptoms or making the injury, pathology, or condition more tolerable to the patient, slowing in the rate of degeneration or decline, making the final point of degeneration less debilitating, improving a subject's physical or mental well-being, or prolonging the length of survival.
  • the treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of a physical examination, neurological examination, and/or psychiatric evaluations.
  • treating cancer and “treatment of cancer” mean to decrease, reduce, or inhibit the replication of cancer cells; decrease, reduce or inhibit the spread (formation of metastases) of cancer; decrease tumor size; decrease the number of tumors (i.e. reduce tumor burden); lessen or reduce the number of cancerous cells in the body; prevent recurrence of cancer after surgical removal or other anti-cancer therapies; or ameliorate or alleviate the symptoms of the disease caused by the cancer.
  • Effective amount and “therapeutically effective amount” are used interchangeably herein, and refer to an amount of an antibody or functional fragment thereof, as described herein, effective to achieve a particular biological or therapeutic result such as, but not limited to, the biological or therapeutic results disclosed herein.
  • a therapeutically effective amount of the antibody or antigen-binding fragment thereof may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or functional fragment thereof to elicit a desired response in the individual. Such results may include, but are not limited to, the treatment of cancer, as determined by any means suitable in the art.
  • isolated can refer to a nucleic acid or polypeptide that is substantially free of cellular material, bacterial material, viral material, or culture medium (when produced by recombinant DNA techniques) of their source of origin, or chemical precursors or other chemicals (when chemically synthesized).
  • an isolated compound refers to one that can be administered to a subject as an isolated compound; in other words, the compound may not simply be considered “isolated” if it is adhered to a column or embedded in an agarose gel.
  • an “isolated nucleic acid fragment” or “isolated peptide” is a nucleic acid or protein fragment that is not naturally occurring as a fragment and/or is not typically in the functional state.
  • the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
  • FIG. 1 Illustration of one method for production of monoclonal antibodies toward the Extracellular Domain (ECD) of CTLA4.
  • ECD Extracellular Domain
  • Antigen, ECD is injected into Balb/c mice.
  • the natural immune system of the mouse develops antibodies against ECD.
  • B-cell rich spleen cells are harvested and fused with immortal SP20 myeloma cells using polyethylene glycol (PEG). Cells are selected based on their ability to make the desired antibody and clones are propagated.
  • Hybridoma clones can be grown in mass to produce large amounts of antibody which can then be purified or injected into a balb/c mouse and the resulting antibody rich ascetic fluid collected.
  • FIG. 2 Illustrates one methods for large scale purification for the isolation of monoclonal antibodies.
  • Antibody rich hybridoma media is run through a protein G sepharose bead column.
  • Antibody bound beads are washed to increase purity and eluted to release 8-10 mg of purified monoclonal antibody.
  • FIG. 3 Anti-CTLA4 hybridoma cell lines produced from immunization with (A) ECD or (B) peptide sequence were isotyped.
  • FIG. 6 ECD and control (Ctrl) protein were spotted onto PVDF membrane in decreasing concentrations from left to right. Western blot analysis was performed with monoclonal antibodies at 1:1000 dilution. Representative dot blots shown.
  • FIG. 7A-7B Detection of CTLA4 in Jurkat cell line by Flow Cytometry.
  • Jurkat cells transfected with human CTLA-4 was stained with either (A) ECD derived monoclonal antibodies or (B) peptide derived monoclonal antibodies followed by Alexa Fluor conjugated anti-mouse IgG.
  • FIG. 8 In Vitro analysis of anti-CTLA4 mABs.
  • Human Jurkat cells were either unactivated (lane a) or activated by the addition of 10 ⁇ g anti-CD3 mAB plus 250 ng purified B7.1 protein (lane b-j). Additionally cells (lanes c-j) received 150 ng recombinant human CTLA-4 protein (rhCTLA4). 10 ⁇ g of the indicated antibodies were used to neutralize the suppressive effects of rhCTLA4 (lanes d-j) for 24-hours in the presence of activating proteins mentioned above. Cell media was harvested and assayed for IL-2 secretion by Elisa.
  • FIG. 9 In Vivo Tumor Suppression by Novel Monoclonal Antibodies.
  • Murine EMT-6/P cells were implanted s.c. in female Balb/c mice. Therapies began when tumors were 50 mm 3 ; Mice received control PBS (i.p.), CTLA-4 antibodies or a combination of anti-CTLA4 (4D11A8 and 5D3H5).
  • One group received anti-CTLA4 (9H10) therapy as a first line treatment and then a second line therapy consisting of clone 5D3H5 antibody.
  • FIG. 10 PD-1 Indirect Elisa to Determine Antibody Titer.
  • FIG. 11A-11C Purified PD1 Antibodies Separated by SDS-PAGE & Stained with Coomassie Brilliant Blue.
  • FIG. 12 Dot Blot Testing of PD-1.
  • FIG. 13 Flow Cytometry Analysis of PD-1 Clones.
  • A Cells only.
  • B Secondary.
  • C Anti-PD1 commercial.
  • D Clone 2A6E6.
  • E Clone 2H11D3.
  • F Clone 3H3F4.
  • G Clone 5B3D5.
  • H Clone 1007D3.
  • FIG. 14 BTLA Indirect Elisa to Determine Antibody Titer.
  • FIG. 15 Dot Blot Testing of BTLA Clones.
  • Immune tolerance allows cancer cells to avoid recognition and elimination by the host immune system.
  • Embodiments of the invention described herein provides methods and compositions for neutralizing or blocking immune suppression.
  • affinity reagents to components of the immune suppression pathways that reduce, attenuate, or block immune suppression are produced and formulated for administration to a patient in need.
  • CTLA4 is a member of the CD28 receptor family along with the co-inhibitory receptors (CIR) PD1 and BTLA, which function by recruiting phosphatases to reverse events activated by phosphorylation.
  • CIR co-inhibitory receptors
  • PD1 ligand (PD-L1 and PD-L2) are B7 family proteins comprised of tandem V-set and C1-set IgSF domains.
  • PD-L1 binds B7-1, one of the ligands of CD28 and CTLA-4, potentially interlocking the PD-1 and CD28/CTLA-4 signaling pathways.
  • Structures of mouse PD-1 complexed with human PD-L1 and mouse PD-L2 revealed that these proteins interact largely orthogonally via their GFCC′C′′ ⁇ -sheets.
  • Embodiments described herein are directed to antibodies or antibody fragments and methods of using the same. Certain aspects are directed to conformation specific or monoclonal antibodies toward cytotoxic T-lymphocyte associated protein-4 (CTLA4) or other CIRs (e.g., PD1 and BTLA), to activate the natural immune response and dampen the progression of cancer directly or indirectly. Thus, cancer can be treated with the administration of such antibodies promoting the death of tumor cells.
  • CTL4 cytotoxic T-lymphocyte associated protein-4
  • BTLA cytotoxic T-lymphocyte associated protein-4
  • a conformation specific antibody binds a protein in its folded conformation and does not bind a protein that has been denatured or unfolded in the region the antibody binds.
  • Confirmation specific antibodies can be produced by immunizing with whole proteins or appropriately folded fragments, e.g., extracelluar domain (ECD) and the like ( FIG. 1 ), Conformation specific antibodies bind to secondary or tertiary structure, as compared to linear epitopes (peptides) to develop therapeutic monoclonal antibodies.
  • ECD extracelluar domain
  • FIG. 1 Conformation specific antibodies bind to secondary or tertiary structure, as compared to linear epitopes (peptides) to develop therapeutic monoclonal antibodies.
  • Certain embodiments are directed to the treatment of cancer by administering an anti-CIR blockade that promotes the death of tumor cells.
  • the anti-CIR blockade can comprise one or more antibodies.
  • the antibodies can be conformation specific antibodies, monoclonal antibodies, or recombinant antibodies.
  • the antibody upon binding its target will disrupt the target's ligand binding interface. In any event, regardless of the mechanism the antibodies will neutralize or block the activity of the target to a sufficient degree to impart a therapeutic response.
  • antibodies can be produced based on the functional and structural domains of the CIR protein(s).
  • Various domains of the targets are used to generate antibodies with therapeutic activity. In certain aspects the following domains were targeted:
  • CTLA4 is a member of the immunoglobulin superfamily, which is expressed on the surface of Helper T cells and transmits an inhibitory signal to T cells.
  • CTLA-4 is similar to the T-cell co-stimulatory protein, CD28, and both molecules bind to CD80 and CD86, also called B7-1 and B7-2 respectively, on antigen-presenting cells.
  • CD80 and CD86 also called B7-1 and B7-2 respectively, on antigen-presenting cells.
  • CD80 and CD86 also called B7-1 and B7-2 respectively, on antigen-presenting cells.
  • CD80 and CD86 also called B7-1 and B7-2 respectively
  • CTLA-4 the anti-CDR3 loop (MYPPPY) was targeted, in particular the amino acid sequence YICKVELMYPPPYYLGIGNGTQI.
  • Another region of CTLA-4 targeted is the C strands, in particular the amino acid sequence EVRVTVLRQADSQVTEVCAATYM, or GKATEVRVTVLRQAD.
  • Still another segment targeted is the C′ strand, in particular the amino acid sequence VTEVCAATYMMGNELTFL.
  • the CTLA-4 dimer interface (comprising residues 51, 53, 59, 106, and 117 of SEQ ID NO:1) is targeted.
  • CTLA-4 and CD28 family residues involved in the CTLA/B7.2 binding site shows greater than 50% conservation across human, murine, rat and rabbit.
  • Analysis of the CTLA-4 and sB7.1 receptor-ligand binding interface shows that the CDR3 loop (MYPPPY) of hCTLA-4 is buried in the concave depression of sB7.1.
  • the ribbon diagram of CTLA-4 dimer shows that the interface is made up of residues from C-terminal to G strand, centered around the A′ strand.
  • human Jurkat leukemia cells which produce and secrete IL-2, represents a marker of T-cell activation.
  • a test was performed to determine the in vitro neutralization capabilities of these CTLA4 monoclonal antibodies.
  • human Jurkat cells responded favorably to activation with anti-CD3 and B7.1 as well as inhibitory signals driven by the addition of recombinant human CTLA-4 (rhCTLA-4) as measured by IL2 release.
  • rhCTLA-4 recombinant human CTLA-4
  • the various clones were competent to inhibit CTLA-4 activity: 3H4E2, 3H4H5, 4D11C9 and 5C4H2 quite effectively. For this this assay, the latter clones showed neutralizing capability similar to a commercially available 9H10 antibody. Clones 4D11C9 and 5D3H5 were also effective.
  • mice 4D11A8, 5C4H2, 5D3H5 and 3H4E2 were chosen for in vivo screening and display of anti-tumor activity. Briefly, female Balb/c mice were injected subcutaneously with mouse EMT-6/P cells. Treatment began once tumors reached 50 mm 3 following intraperitoneally (i.p.) delivery. Mice that received a control treatment (PBS) showed rapid tumor growth as compared to clones 4D11A8, 5C4H2 or 3H4E2. In comparison, mice treated with anti-CTLA-4 (9H10), clone 5D3H5 or a combination of 5D3H5 plus 4D11A8 responded to therapy ( FIG. 9 ).
  • Certain embodiments are directed to antibodies that specifically bind CIRs PD1 or BTLA.
  • PD1 Programmed cell death protein 1 or CD279
  • CD279 is a cell surface receptor that belongs to the immunoglobulin superfamily and is expressed on T cells and pro-B cells.
  • PD-1 binds two ligands, PD-L1 and PD-L2.
  • PD-1 and its ligands play a role in down regulating the immune system by preventing the activation of T-cells.
  • BTLA B- and T-lymphocyte attenuator or CD272
  • BTLA B- and T-lymphocyte attenuator or CD272
  • BTLA interacts with a B7 homolog, B7H4.
  • BTLA displays T-Cell inhibition via interaction with tumor necrosis family receptors (TNF-R), not just the B7 family of cell surface receptors.
  • TNF-R tumor necrosis family receptors
  • the PD1 and BTLA antibodies disrupt co-inhibitory receptor/ligand binding interface and neutralize or block the activity of PD1 or BTLA, respectively.
  • Antibodies to PD1 (see FIG. 10 to FIG. 14 ) and BTLA (see FIG. 14 and FIG. 15 ) are developed based on the functional and structural domains of the CIR proteins. The following domains were targeted to develop therapeutic PD1 and BTLA antibodies:
  • PD1 antibodies are developed using the amino acid sequence of the C′ strand, in particular SESFVLNWYRMSPS.
  • the C′C′′ strand is also targeted, in particular QTDKLAAFPEDRSQPGQDC.
  • Another target of PD1 is the FG strand, in particular DSGTYLCGAISLAPKAQIKES.
  • the G strand is also targeted, in particular KAQIKESLRAELRVTER. Residues were identified from the GFC′C′′ strands and C′C′′, and FG loops of PD-1 contribute to the binding interface of PDL2.
  • BTLA antibodies are developed using the amino acid sequence of the A′ strand, in particular DVQLYIKRQSEHSILA. G° strand CSANFQSNLIESHS. CC′ loop, RPHVTWCKLNGTTCVK. These sites were selected based on the information regarding BTLA/ligand binding. Crystollography coupled to mutagenesis studies show that strands A′ and G° are part of the BTLA/ligand binding interface. Unlike other CD28 family members, the BTLA binding surface is located along the edge of the I-set Ig domain.
  • BTLA blockade may potentially improve T cell antitumor immunity.
  • the invention also provides compositions comprising one or more anti-cancer agents, e.g., therapeutic antibodies, with one or more of the following: a pharmaceutically acceptable diluent; a carrier; a solubilizer; an emulsifier; a preservative; and/or an adjuvant.
  • Such compositions may contain an effective amount of at least one anti-cancer agent.
  • the use of one or more anti-cancer agents that are provided herein in the preparation of a pharmaceutical composition of a medicament is also included.
  • Such compositions can be used in the treatment of a variety of cancers. In certain embodiments the treatment is for leukemia or breast cancer.
  • the anti-cancer agents may be formulated into therapeutic compositions in a variety of dosage forms such as, but not limited to, liquid solutions or suspensions, tablets, pills, polymeric microcapsules or microvesicles, liposomes, and injectable or infusible solutions.
  • dosage forms such as, but not limited to, liquid solutions or suspensions, tablets, pills, polymeric microcapsules or microvesicles, liposomes, and injectable or infusible solutions.
  • the preferred form depends upon the mode of administration and the particular disease targeted.
  • the compositions also preferably include pharmaceutically acceptable vehicles, carriers, or adjuvants, well known in the art.
  • compositions may contain components for modifying, maintaining, or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or penetration of the composition.
  • Suitable materials for formulating pharmaceutical compositions include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite); buffers (such as acetate, borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents;
  • compositions can be administered using conventional modes of delivery including, but not limited to, intravenous, intraperitoneal, oral, subcutaneous, intraarterial, intramuscular, intrapleural, and intrathecal administration.
  • administration can be by perfusion through a regional catheter. Local administration to a tumor is also contemplated.
  • the administration may be by continuous infusion or by single or multiple boluses.
  • the anti-cancer agents may be administered in a pyrogen-free, parenterally acceptable aqueous solution.
  • composition of the invention may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder.
  • Such formulations may be stored either in a ready-to-use form or in a form (e.g., lyophilized) that is reconstituted prior to administration.
  • stabilizers can be used, such as sucrose, trehalose, or glycine. Typically, such stabilizers will be added in minor amounts ranging from, for example, about 0.1% to about 0.5% (w/v).
  • Surfactant stabilizers such as TWEEN®-20 or TWEEN®-80 (ICI Americas, Inc., Bridgewater, N.J., USA), may also be added in conventional amounts.
  • compositions intended for in vivo use are usually sterile. To the extent that a given compound must be synthesized prior to use, the resulting product is typically substantially free of any potentially toxic agents.
  • compositions for parental administration are also sterile, substantially isotonic and made under GMP conditions.
  • such doses are between about 0.001 mg/kg and 1 mg/kg body weight, preferably between about 1 and 100 ⁇ g/kg body weight, most preferably between 1 and 10 ⁇ g/kg body weight.
  • Therapeutically effective doses will be easily determined by one of skill in the art and will depend on the severity and course of the disease, the patient's health and response to treatment, the patient's age, weight, height, sex, previous medical history and the judgment of the treating physician.
  • the cancer cell is in a patient.
  • the patient may or may not have a solid tumor.
  • embodiments may further involve performing surgery on the patient, such as by resecting all or part of the tumor.
  • Compositions may be administered to the patient before, after, or at the same time as surgery.
  • patients may also be administered directly, endoscopically, intratracheally, intratumorally, intravenously, intralesionally, intramuscularly, intraperitoneally, regionally, percutaneously, topically, intrarterially, intravesically, or subcutaneously.
  • Therapeutic compositions may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more times, and they may be administered every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, or 1, 2, 3, 4, 5, 6, 7 days, or 1, 2, 3, 4, 5 weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months.
  • Methods of treating cancer may further include administering to the patient chemotherapy or radiotherapy, which may be administered more than one time.
  • Chemotherapy includes, but is not limited to, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, bisulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen, taxotere, taxol, transplatinum, 5-fluorouracil, vincristin, vinblastin, methotrexate, gemcitabine, oxaliplatin, irinotecan, topotecan, or any analog or derivative variant thereof.
  • CDDP cisplatin
  • carboplatin carboplatin
  • procarbazine
  • Radiation therapy includes, but is not limited to, X-ray irradiation, UV-irradiation, ⁇ -irradiation, electron-beam radiation, or microwaves.
  • a cell or a patient may be administered a microtubule stabilizing agent, including, but not limited to, taxane, as part of methods of the invention. It is specifically contemplated that any of the compounds or derivatives or analogs, can be used with these combination therapies.
  • the cancer that is administered the composition(s) described herein may be a bladder, blood, bone, bone marrow, brain, breast, colorectal, esophagus, gastrointestine, head, kidney, liver, lung, nasopharynx, neck, ovary, pancreas, prostate, skin, stomach, testicular, tongue, or uterus cell.
  • the cancer is blood (e.g., lymphoma, leukemia, etc.) or breast cancer.
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PL3303394T3 (pl) 2015-05-29 2020-11-16 Agenus Inc. Przeciwciała anty-ctla-4 i sposoby ich zastosowania
WO2017011580A2 (fr) 2015-07-13 2017-01-19 Cytomx Therapeutics, Inc. Anticorps anti-pd-1, anticorps anti-pd-1 activables, et leurs procédés d'utilisation
BR112018003186A2 (pt) 2015-09-01 2018-09-25 Agenus Inc. anticorpos anti-pd-1 e seus métodos de uso
WO2018035710A1 (fr) 2016-08-23 2018-03-01 Akeso Biopharma, Inc. Anticorps anti-ctla4
JP6992068B2 (ja) 2016-12-07 2022-02-03 アジェナス インコーポレイテッド 抗ｃｔｌａ－４抗体およびそれらの使用方法
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CN112203681A (zh) * 2018-02-07 2021-01-08 免疫基因有限公司 疫苗组合物及其用途
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